Remedy for degenerative intervertebral discs

ABSTRACT

To treat diseases accompanied by degenerative intervertebral discs, in particular, disk herniation, lumbar pain, discopathy and osteoarthritits of the spine, it is intended to provide a drug for administering a human-origin protease directly to an affected part of such a disease accompanied by degenerative intervertebral discs. As the human-origin protease, use can be made of MMP-3, MMP-7, etc.

TECHNICAL FIELD

The present invention relates to an agent for treating diseasesassociated with degenerative intervertebral disc which comprises ahuman-derived protease as an active ingredient. The agent is directlyadministered to the affected part of a disease associated withdegenerative intervertebral disc, and is effective for disc herniation,lumbar pain, discopathy and spondylosis and the like, by promotingnatural resorption of the herniated disc.

BACKGROUND ART

Intervertebral discs, the disc-shaped cartilaginous bodies interposedbetween adjacent vertebrae, are each composed of an outer annulusfibrosus surrounding the inner nucleus pulposus, and comprisecartilaginous components such as proteoglycans, aggrecans, type IIcollagen and the like. Intervertebral discs lose their elasticity due toreduction of water content in discs occurring primarily as a result ofaging, with concomitant loss of cartilaginous components and build-up offibrous tissue, which leads to destruction of the double structure ofthe inner nucleus pulposus and the outer annulus fibrosus. This changeis referred to as “degenerative intervertebral disc”. Degenerativeintervertebral disc progresses with increasing age beginning in thethird decade, with degeneration of virtually all intervertebral disctissue occurring by age 70. Herniated disc is caused when degenerativenucleus pulposus ruptures the fragile fibrous tissue and protrudes intothe spinal canal, compressing against the nerve root and cauda equinaand producing pain or paralysis. Lumbago can also be caused by abnormalbone metabolism, external injury, tumor or the like, but usually resultsfrom degeneration of intervertebral discs.

Magnetic Resonance Imaging (MRI) is widely used for diagnosis ofdegenerative intervertebral discs. Because it does not involve exposureas with X-rays, it can be used for frequent examination. Sequentialimaging has confirmed a natural resorption mechanism whereby the volumeof herniated discs decreases with time. Also, use of the contrast agentgadolinium-diethylenetriamine pentaacetic acid (Gd-DTPA) has shownabundant neovascularization in herniated discs when an imaging effect isseen in the herniated intervertebral disc tissue, suggesting that morenatural resorption is occurring. This principle can be utilized fordiagnosis and prognosis.

Immunohistological examination of extracted human surgical samples ofherniated disc masses has revealed increased neovascularization andinfiltration of numerous inflammatory cells consisting primarily ofmacrophages, in the cartilage matrix of herniated disc masses (Haro etal., Spine 21(1996), 1647-1652). It has been confirmed that theinfiltrating macrophages and intervertebral disc chondrocytes abundantlyexpress the matrix metalloproteinases (MMPs) MMP-3 and MMP-7 (Haro etal., Spine 22(1997), 1098-1104 and Haro et al. J. Spinal. Disorders12(1999), 245-249). MMPs are enzymes which function mostly in theneutral range and are physiologically expressed intraarticularly and inthe intervertebral disc tissue. Proteoglycans and aggrecans, majorcartilage tissue components, are the substrates of MMP-3 and MMP-7. Itis therefore assumed that MMP-3 and MMP-7 play important roles in thedegradation and resorption of herniated discs.

When wild-type mouse intervertebral discs are co-cultured with activemacrophages, reduction in intervertebral disc mass is greater than withco-culturing of macrophages and intervertebral discs derived from MMP-3deficient mouse. Experiments using wild-type mice and MMP-3 deficientmice have confirmed that MMP-3 has a chemotactic effect on macrophagescausing them to migrate toward the intervertebral discs (MMP-3 functionsas a chemotactic factor for macrophages) (Haro et al., J. Clin. Invest.105(2)(2000), 133-141).

MMP-7 elicits the inflammatory cytokine TNF-α, and TNF-α has beenreported to promote production of MMP-3 in intervertebral disc cells(Haro et al., J. Clin. Invest. 105(2)(2000), 143-150). However, it hasnever been attempted to use human-derived proteases such as MMP-3 andMMP-7 expressed in human herniated discs as therapeutic agents.

Surgical treatment for herniated discs ordinarily attempts to achievenerve decompression by removal of the herniated disc mass. However,because the condition is common among adolescent and middle age groupsand relatively frequent among sport athletes, a non-invasive treatmenthas been sought in order to avoid surgery.

Treatment by injection of enzymes such as plant-extracted chymopapaininto herniated discs is already practiced in the U.S. and Europe, butimmunoreaction and neurotoxicity have been reported. Also, although ithas been reported that injection of chymopapain into herniated discsites gradually restores herniated disc cavities produced byproteolysis, this is attributed to hyperplasia not of the cartilaginousmatrix but of fibrous connective tissue. Histological observations ofchymopapain-injected canine discs have reportedly revealed replacementof the nucleus pulposus center with fibrocartilaginous tissue (Kudo etal., J. Vet. Med. Sci. 1993, April, 55(2) 211-5). According toexperimentation by the present inventors, cases where chymopapain wasinjected into canine herniated discs showed degradation of thecartilaginous matrix throughout the entire nucleus pulposus and annulusfibrosus, with the surviving chondrocytes markedly reduced andextensively damaged. Thus, lysis of herniated disc with chymopapain isbelieved to either reduce or eliminate intervertebral disc regenerativecapacity. Since chondrocytes maintain their function while suspended inthe matrix, the matrix is indispensable for intervertebral discregeneration. Consideration of these publicly known facts together withknowledge confirmed by the present inventors suggests that in theconventional methods in which proteases are directly administered toherniated disc sites for removal of degenerative nucleus pulposus, it isdifficult to sustain the matrix which is required to support theintervertebral disc regenerative capacity of chondrocytes.

DISCLOSURE OF THE INVENTION

Surprisingly, it was discovered that injection of MMPs, types ofhuman-derived proteases, into herniated discs promotes resorption ofherniated discs but does not injure normal chondrocytes. Specifically,it was possible to degrade herniated discs while maintainingintervertebral disc regenerative capacity. As mentioned above, thematrix is absolutely essential for maintaining chondrocyte function. Thepresent invention is based on the revolutionary knowledge that injectionof MMPs, which are human-derived proteases, into herniated disc promotesselective resorption of the herniated disc while preserving normalchondrocytes. The invention provides a completely novel herniated disctreatment which does not entail the risk of injury to normalchondrocytes as occurs when using hitherto known chymopapain.

It is an object of the present invention to provide an agent fortreating diseases associated with degenerative intervertebral disc whichcomprises as an active ingredient a human-derived protease believed tocontribute to natural human herniated disc resorption, and which isdirectly administered to the degenerative intervertebral disc. Theinvention further provides a treatment method for disorders associatedwith degenerative intervertebral disc whereby the human-derivedproteases are directly administered to the degenerative intervertebraldisc. The target disorders for the treatment of the invention areconditions associated with degenerative intervertebral disc and includeherniated disc, lumbago, discopathy and spondylosis.

The aforementioned MMP-3 and MMP-7 may be mentioned as substancesbelieved to contribute to resorption of herniated disc based on researchto date. A similar effect is thought to be exhibited by MMP-8, MMP-13,MT1-MMP (MMP-14) and aggrecanase. MMP-3 (E.C.3.4.24.17), also known asstromelysin-1 or transin, degrades fibronectin, laminin, proteoglycanand various collagens. MMP-7 (E.C.3.4.24.33), also known as matrilysinor PUMP, degrades type IV and type X collagen, elastin, fibronectin,gelatin, laminin and proteoglycan. MMP-8 (E.C.3.4.24.34), also known ascollagenase-2 or neutrophil collagenase, degrades type I collagenpreferentially over type II or type III collagen. MMP-13 is also knownas collagenase-3 and is highly specific for type II collagen. MT1-MMP(membrane type-1 matrix metalloproteinase), also known as MMP-14,activates pro-MMP-2 and pro-MMP-13. Aggrecanase belongs to the ADAM-TS(a disintegrin and metalloproteinase-thrombospondin) family and degradescartilage aggrecan.

These proteases have recently been produced and provided by recombinanttechnology. The present inventors have utilized and confirmed theefficacy of these proteases by injection into conditions associated withdegenerative intervertebral disc, such as herniated disc. In addition,it was confirmed that these human-derived proteases do not carry therisk of injuring normal chondrocytes which is exhibited by chymopapain,and the present invention was thereupon completed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pair of bar graphs showing changes in wet weight ofherniated disc specimens cultured with MMP-3, MMP-7, mixtures thereof,chymopapain as a positive control and a control (DMEM medium).

FIG. 2 is a set of photographs showing the results of Safranin Ostaining of tissue samples obtained by culturing herniated discspecimens with MMP-3, MMP-7, mixtures thereof and chymopapain as apositive control.

FIG. 3 is a set of photographs showing the results of Safranin Ostaining of vertebral samples prepared by injecting MMP-3, MMP-7,chymopapain as a positive control and normal saline (NS) into rabbitintervertebral discs and sacrificing after one week.

FIG. 4 is a micrograph of a herniated disc tissue sample(hematoxylin-eosin stained) extracted from spontaneous herniated discfrom a miniature dachshund.

FIG. 5 is a fluoroscopic image showing beagle spontaneous herniated discupon injection of MMP-7.

FIG. 6 is a pair of images from MRI (lipid T1-weighted) before (Pre) andafter (Post) injection of MMP-7 into beagle spontaneous herniated disc.

FIG. 7 is a pair of images from MRI (water T2-weighted) before (Pre) andafter (Post) injection of MMP-7 into beagle spontaneous herniated disc.

FIG. 8 is a pair of images from MRI (water T2-weighted) before (Pre) andafter (Post) injection of MMP-3 into beagle spontaneous herniated disc.

FIG. 9 is a photograph showing cut vertebral samples prepared byinjecting MMP-7 and a normal saline as a control into beagle spontaneousherniated discs, and sacrificing after one week.

FIG. 10 is a pair of photographs showing the results of Safranin Ostaining of vertebral samples prepared by injecting MMP-7 and normalsaline as a control into beagle spontaneous herniated discs, and thensacrificing after one week; the results are shown for injection of MMP-7and injection of normal saline.

FIG. 11 is a photograph showing the results of Safranin O staining of avertebral sample prepared by injecting MMP-7 into beagle spontaneousherniated disc, and sacrificing after one week.

FIG. 12 is a photograph showing the results of Safranin O staining of avertebral sample prepared by injecting normal saline as a control intobeagle spontaneous herniated disc, and sacrificing after one week.

FIG. 13 is an image showing the results of a Western blot for testing ofproteoglycan degradation by MMP-7.

FIG. 14 is an image showing the results of Safranin O staining of apathological sample prepared by injecting chymopapain into canineherniated disc, and sacrificing after one week after injection.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention provides an agent for treating diseases associatedwith degenerative intervertebral disc which comprises as an activeingredient one or more human-derived proteases and is administereddirectly to the site of the condition associated with degenerativeintervertebral disc. In particular, it is an agent for treatingherniated disc, lumbago, discopathy and spondylosis. The proteases asthe active ingredients are extracellular matrix proteases such as, forexample, MMP-3, MMP-7, MMP-8, MMP-13, MT1-MMP (MMP-14) and aggrecanase.

The agent for treating diseases associated with degenerativeintervertebral disc according to the invention is directly administeredto, for example, a herniated disc site. The method of administering theagent of the invention may be administration into the intervertebraldisc near the hernia. In disc puncture, for example, a puncture needleis advanced subcutaneously under fluoroscopic control. The innercylinder is then advanced in the direction of the protruded disc forselective puncture of the protruded herniated disc, and the drug agentof the invention is injected. It may also be administered by epiduralspace injection. Administration of the agent of the invention in thismanner can promote natural resorption of the herniated disc.

The use of an agent for treating diseases associated with degenerativeintervertebral disc according to the invention requires diagnosiswherein a neural symptom such as radiculopathy, cauda equina syndrome ormyelopathy is observed and the condition of nerve compression and thesite and severity of the degenerative intervertebral disc, such asherniated disc, is confirmed by magnetic resonance imaging (MRI).Discographic examination is also important for imaging of the herniateddisc mass at the site to be treated, in order to accurately judge thesite and extent of the herniated disc.

Thus, one mode of the invention is a treatment method for herniated discwherein a patient suspected of having a degenerative intervertebraldisc, such as a herniated disc, is examined by MRI and discography and,if the patient is found to have a degenerative intervertebral disc suchas a herniated disc, a human-derived protease of the invention isdirectly administered to the herniated disc site as appropriate for thepathology, to promote natural resorption of the herniated disc tissue.

The dosage of the agent for treating diseases associated withdegenerative intervertebral disc according to the invention will differdepending on the severity of the degenerative intervertebral disc, suchas herniated disc, the condition of the patient, the type of protease,etc. The agent for treating diseases associated with degenerativeintervertebral disc according to the invention is administered directlyto the affected site, and its dosage will normally be in the range ofabout 1 μg to 100 mg and preferably 100 μg to 1 mg per administration.The frequency of administration is preferably from once to severaltimes, and may be increased depending on the degree of resorption.

EXAMPLES

The present invention will now be explained in greater detail by thefollowing examples, with the understanding that the invention is in noway limited by these examples.

Example 1 Organ Culturing Experiment Using Human Surgical Herniated DiscSpecimen

A surgically extracted herniated lumbar disc specimen (stored at 4° C.)was finely sliced, and the wet weight was measured for dispensation intospecimen portions of 60-80 μg, which were placed in a 96-well plate. Thesample solutions described below were added and culturing was carriedout for 24 hours in a CO₂ incubator at 37° C.

(1) Control/DMEM medium 200 μl

(2) Human recombinant MMP-3 (45 kDa) 20 μg/200 μl

(3) Human recombinant MMP-7 (19 kDa) 20 μg/200 μl

(4) Human recombinant MMP-3 10 μg+human recombinant MMP-7 10 μg/200 μl

(5) Human recombinant MMP-3 20 μg+human recombinant MMP-7 20 μg/200 μl

(6) Chymopapain 1, 5, 10, 50, 100, 500 pkt (picoKatal)/200 μl

The human recombinant MMP-3 (CHEMICON) and human recombinant MMP-7(CHEMICON) were dissolved in DMEM medium at 4° C. The chymopapain wasused as a positive control, dissolving chymopapain (ICN Biomedicals) ina solution containing 1 mM EDTA, 0.067 mM mercaptoethanol and 5.5 mMcysteine hydrochloride, and carrying out incubation at 25° C., pH 6.2for 30 minutes for activation prior to addition to the DMEM medium.

The wet weight of the obtained culture product was measured andstatistical calculation was performed. The results are shown in FIG. 1.As clearly seen in FIG. 1, Using MMP-3, MMP-7 or both in combinationsignificantly reduced the wet weight compared to the control.

Next, the specimen was placed in 4% paraformaldehyde to prepare a tissuesample which was then stained with Safranin O, giving the results shownin FIG. 2. As a result, the cultured organs with addition of MMP-3,MMP-7 or chymopapain exhibited reduction in staining (resorption ofherniated disc) which was superior to the control.

Example 2 Experiment for Injection into Rabbit Intervertebral Disc

Japanese white rabbits (3-4 kg) were treated by the procedure describedbelow, and the effects of intervertebral disc injection of drug agentswere examined.

1. Premedication treatment was subcutaneous injection of 0.5-1.0 ml/kgof ketamine hydrochloride.

2. The bodies were fixed, and anchored with tape on a line along the earvein with a 23 G winged needle. Pentobarbital sodium was diluted tohalf-concentration (0.5 mg/ml) with normal saline and 0.5-1.0 ml/kg wasinjected in one shot for intravenous anesthesia. Appropriate 1 mlportions were then injected thereafter. The bodies were then shaved witha hair clipper and fixed in the lateral prostrate position (posteriorapproach).

3. A draping and electric scalpel (air tome) set was used to cut theskin, the subcutaneous area was opened up, and the muscle was separatedfrom the vertebral body and costa to expose the intervertebral discs.

4. The following drug agents were directly injected into theintervertebral discs.

(1) Human recombinant MMP-3 10 μg/100 μl and 40 μg/100 ηl (concentratedwith Microcon YM-3)

(2) Human recombinant MMP-7 10 μg/100 μl and 40 μg/100 μl (concentratedwith Microcon YM-3)

(3) Chymopapain 1 kpt and 5 kpt

(Activated by dissolution in solution containing 1 mM EDTA, 0.067 mMmercaptoethanol and 5.5 mM cysteine hydrochloride, and incubation at 25°C., pH 6.2 for 30 minutes)

(4) Control: Normal saline (NS)

A 100 μl portion of the drug agent was injected into the intervertebraldisc. Different concentrations of the drug agent were administered attwo locations of the intervertebral disc, and the normal saline alonewas administered at a single location of the intervertebral disc, afterwhich a marking wire was embedded in the vertebral body.

5. Each layer was closed off with a nylon needle and the rabbits werefreed within their cages.

6. One week after injection, 3 ml of ketamine hydrochloride wasinjected, and after ensuring the line, a 23 G winged needle was fixed atthe ear vein, and 3 ml of pentobarbital sodium (1 mg/ml) wasadministered (total: 5-7 ml).

7. The vertebral body was extracted en bloc and fixed with a 4% formalinsolution to prepare a tissue sample.

8. The tissue sample was deparaffinized and stained for 5 minutes with0.03% Fast Green dissolved in 1% aqueous acetic acid. After washing with1% aqueous acetic acid, it was further stained for 7 minutes with 0.25%Safranin O.

The results are shown in FIG. 3. The MMP-3, MMP-7 and chymopapaininjected groups had significantly reduced staining compared to thecontrol group. The MMP-3 injected group also produced intervertebraldisc narrowing.

Example 3 Experiment for Injection into Canine Herniated Disc

Dogs, and particularly beagles, have a high incidence of herniated disc,exhibiting paralysis of both of the lower limbs in severe cases. Thehistology of canine herniated disc is highly similar to the histology ofhuman herniated disc. A sample was prepared from tissue surgicallyextracted from the 1st/2nd lumbar herniated disc of a 7-year-old,6.35-kg miniature dachshund that had suffered herniated disc and lowerlimb paralysis, and was hematoxylin-eosin stained and imaged on amicrograph, shown in FIG. 4. This tissue sample demonstrates that canineherniated disc is highly suitable as a model for human herniated disc.

Drug agents were injected into the herniated discs of beagles withspontaneous herniated disc, and the effects were examined in thefollowing manner. A 7-year-old, 12.75-kg beagle was used for a humanrecombinant MMP-7 test, and a 7-year-old, 14.4-kg beagle was used for ahuman recombinant MMP-3 test. The disc was imaged by MRI beforeinjection of the drug agent, to confirm normal disc height, height ofdegeneration and height of herniated disc.

Administration of MMP-7: The beagle was injected with 20 μg/200 μl intothe disc between the 12th thoracic vertebra (T12) and 13th thoracicvertebra (T13), and with 10 μg/100 μl into the disc between the 13ththoracic vertebra (T13) and 1st lumbar vertebra (L1) and the discbetween the 1st lumbar vertebra (L1) and 2nd lumbar vertebra (L2). As acontrol, 200 μl of normal saline was injected into the disc between the2nd lumbar vertebra (L2) and third lumbar vertebra (L3).

Administration of MMP-3: The beagle was injected with 10 μg/100 μl intothe disc between the 11th thoracic vertebra (T11) and 12th thoracicvertebra (T12), with 20 μg/200 μl into the disc between the 12ththoracic vertebra (T12) and 13th thoracic vertebra (T13), and with 10μg/100 μl into the disc between the 13th thoracic vertebra (T13) and 1stlumbar vertebra (L1). As a control, 200 μl of normal saline was injectedinto the disc between the 10th thoracic vertebra (T10) and 11th thoracicvertebra (T11).

Injection of the agents into the intervertebral discs was accomplishedusing a spinal needle under fluoroscopic control (FIG. 5). The syringeused was a tuberculin syringe (total volume: 1 ml). MRI measurement wasrepeated one week after injection. The MRI images of the discs beforeand after injection are shown in FIGS. 6 to 8.

FIG. 6 is a pair of T1-weighted MRI images showing the results usingMMP-7, and FIG. 7 is a pair of T2-weighted MRI images showing theresults using MMP-7. T1-weighted images are effective for visualizingactual herniated disc, while T2-weighted images are effective forvisualizing resolution of the cartilage tissue of intervertebral discs.The herniated disc observed between T12 and T13 before injection hasclearly undergone resorption in the image after injection. FIG. 8 is apair of water content T2-weighted images showing the results usingMMP-3, in which resorption of the herniated disc due to injection of theagent is observed.

The MMP-7 administered beagle was then sacrificed, and the vertebralunit comprising the injected disc was extracted en bloc, fixed with a 4%paraformaldehyde solution and subjected to a decalcification procedure,after which a tissue sample was prepared (FIG. 9). Specifically, fixingwith the 4% paraformaldehyde solution was followed by rinsing with 10 mMPBS and delipidization with ethanol and chloroform. Next, a decalcifiedsolution (Plank Rychlo method: Wako, Japan) was used for adecalcification procedure. Upon confirming adequate decalcification,paraffin embedding was performed to obtain a tissue sample.

After deparaffinization of the tissue sample, it was stained for 5minutes with 0.03% Fast Green dissolved in 1% aqueous acetic acid. Afterthen washing with 1% aqueous acetic acid, it was further stained for 7minutes with 0.25% Safranin O.

FIG. 10 shows photographs of strips thereof (macro image). The striplabeled “After MMP-7 injection” is a strip from the location injectedwith 200 μl of MMP-7 (12th and 13th thoracic vertebrae), and the striplabeled “After normal saline injection” is a strip taken from betweenthe 2nd and 3rd lumbar vertebrae injected with normal saline. The stripsare shown in greater detail in FIGS. 11 and 12. FIG. 11 shows the stripinjected with MMP-7, together with the same macro image at the bottom ofFIG. 10 shown at the upper left corner. The macro image is shown at 2×magnification at the middle of the page, and a portion of the 2×magnified image is shown at additional 10× magnification at the bottomof the page. FIG. 12 shows the strip injected with normal saline, withthe macro image shown at the upper left corner, 2× magnification at themiddle, and 10× magnification at the bottom. When FIG. 10, FIG. 11 andFIG. 12 are compared, it is seen that injection of MMP-7 significantlyreduced staining compared to normal saline. This indicates that theMMP-7 injection had destroyed the structure of the nucleus pulposus. Incontrast, disturbance of the nucleus pulposus structure with injectionof normal saline was found only at the site of injection, and stainingwas unchanged. Thus, no destruction of the cartilaginous matrix wasfound.

In FIG. 11 showing the results of MMP-7 injection, the nucleus pulposusinjected with MMP-7 appears as a degraded matrix, but normalchondrocytes were preserved in the other nucleus pulposus areas and inthe annulus fibrosus. Thus, it was concluded that the normalchondrocytes retained their function of producing the cartilaginousmatrix and regenerating the intervertebral disc.

Example 4 Molecular Biological Examination Using Canine Herniated Disc

In the same manner as Example 3, a beagle (7-year-old, 14.0 kg bodyweight) with spontaneous herniated disc was injected with 20 μg/200 μlof MMP-7 into the disc between the 2nd cervical vertebra (C2) and 3rdcervical vertebra (C3), and with 200 μl of normal saline into the discbetween the 3rd cervical vertebra (C3) and 4th cervical vertebra (C4).The protein was extracted from the discs one week after injection, andhuman adult cartilage proteoglycan antibody was used for molecularbiological examination of intervertebral disc cartilage degradation byWestern blotting. Cross-reaction of human adult cartilage proteoglycanantibody with canine has been confirmed, and the antibody has beendemonstrated to recognize the core protein and keratan sulfate sidechain.

After sacrificing the beagle, the vertebral unit was immediatelyextracted and stored in ice. Next, the intervertebral tissue at thetarget site was carefully separated using a scalpel or the like to avoidcontamination with the surrounding tissue. The sampled tissue was placedin a 10 cm dish, and then a solution comprising one proteinase inhibitortablet (Roche: proteinase inhibitor cocktail tablet) dissolved in 4 mlof RIPA (Radio Immuno Precipitation Assay) buffer solution was added andcrushed in the dish. The composition of the RIPA buffer solution was 50mM Tris-HCl buffer (pH 8.0) containing 150 mM NaCl, 1% Triton-100, 0.5%sodium deoxycholate and 0.1% SDS. After stirring overnight at 4° C., thesolution in the dish was taken and centrifuged at 4° C., 3000 rpm for 5minutes. The supernatant was used as a protein extract for theexperiment.

As a positive control to contrast with the beagle intervertebral discprotein, there was used protein extracted from normal human kneecartilage obtained during surgical removal of a bone tumor surroundingthe knee joint.

The concentration of each protein extract solution was adjusted byprotein assay, and then Laemmli Sample Buffer was added, the mixture wasboiled at 95° C. for 3 minutes, and SDS-PAGE (80 V, 180 min) was carriedout using 12% gel (PAG Minigel, Daiichi Pure Chemicals). It was thentransferred to a nitrocellulose membrane (0.1 A, 45 min). After thenreacting it with human adult cartilage proteoglycan antibody by Chemicon(Temecula, Calif., USA), the product was colored with an ECL kit(Amersham Pharmacia Biotech, UK). The results are shown in FIG. 13. Inthe photograph, M represents a molecular weight marker, PC representsthe positive control using protein extracted from normal human kneecartilage, C2/3 represents protein extracted from MMP-7 injectedintervertebral disc, C3/4 represents protein extracted from normalsaline injected intervertebral disc, and C5/6 represents proteinextracted from an untreated intervertebral disc (between the 5th and 6thcervical vertebrae).

The bands from 40 kDa to 100 kDa represent the G1 domain-containing coreprotein and keratan sulfate side chains. The lack of one single bandreflects the presence of several decomposition products due toproteoglycan decomposition by physiological enzymes, since the specimenwas a clinical specimen. In the case of the positive control (PC) whichused protein extracted from normal human knee cartilage, variousproteoglycan-degrading enzymes such as MMPs and aggrecanase were presentand therefore proteoglycan decomposition products were detected.Proteoglycan decomposition products were also detected with normalsaline injection (C3/4) and the untreated sample (C5/6). In comparison,MMP-7 injection clearly reduced the amount of proteoglycan decompositionproducts. This result demonstrates that MMP-7 had degraded theproteoglycan in the herniated disc.

The organ culturing experiment using a surgically removed herniated discspecimen (Example 1) clearly demonstrated that herniated disc isdegraded by MMP-3 and MMP-7. Also, the experiment of injection intorabbit intervertebral disc (Example 2), the experiment of injection intobeagle spontaneous herniated disc (Example 3) and the molecularbiological examination using canine herniated disc (Example 4) clearlyshowed that degradation of the intervertebral disc and herniated discmatrix occurs by intervertebral disc injection of MMP-3 and MMP-7. Thesefindings confirmed that MMP-3 and MMP-7 are effective for treatment ofherniated disc.

Reference Example

One week after injection of chymopapain into the herniated disc of acanine, it was sacrificed and a pathological sample was prepared andstained in the same manner as Example 3. Photographs are shown in FIG.14. In this case, the cartilaginous matrix was degraded throughout theentire nucleus pulposus and annulus fibrosus, with very few survivingchondrocytes. Thus, it was concluded that injection of chymopapain intothe site of herniated disc produces injury to normal chondrocytes,thereby reducing the intervertebral disc regenerative capacity incomparison to injection of MMP-7 according to the invention.

INDUSTRIAL APPLICABILITY

The agent for treating diseases associated with degenerativeintervertebral disc according to the invention, whereby a human-derivedprotease such as MMP-3 and MMP-7 is directly administered to the site ofa disease associated with degenerative intervertebral disc, allows suchdiseases to be cured rapidly. The diseases include, for example,herniated disc, lumbago, discopathy and spondylosis. Human-derivedproteases are highly superior to plant-derived enzymes such aschymopapain which have been conventionally applied in the clinic, inthat they do not cause serious immunoreactions such as anaphylaxis, anddo not injure normal chondrocytes.

1-4. (canceled)
 5. A method of therapy for treating diseases associatedwith degenerative intervertebral disc characterized by directlyadministering an active ingredient comprising one or more human-derivedproteases to the affected site of a disease associated with degenerativeintervertebral disc.
 6. The method according to claim 1, wherein thedisease associated with degenerative intervertebral disc is herniateddisc, lumbago, discopathy or spondylosis.
 7. The method according toclaim 1, wherein the human-derived protease is an extracellular matrixprotease.
 8. The method according to claim 1, wherein the activeingredient comprises one or more extracellular matrix proteases selectedfrom the group consisting of MMP-3, MMP-7, MMP-8, MMP-13, MT1-MMP andaggrecanase.